The overall objective is to address the questions of whether a diminished capacity to regulate intracellular pH (pHi) and free cytoplasmic Ca2+ (pCai) at temperatures achievable during local hyperthermia can be correlated with thermal cell death and whether modification of pHi and pCai by specific drugs and other agents can sensitize cells to heat. Results obtained will relate directly to mechanisms by which heat kills tumor cells and the development of thermotolerance and as such has implications for enhancement of clinical local hyperthermia. Regulation of pHi and pCai by V79 fibroblasts in log and plateau phase growth will be examined before, during and after heat treatments (37-45C) and with thermotolerant cells as a function of extracellular pH (pHe) with and without intracellular acid loading. Mitochondrial membrane potential, lysosomal pH and cytoplasmic pHi and pCai will be determined with fluorescent probes and either a conventional fluorescence spectrophotometer or a fluorescence microscope coupled to an intensified photodiode array to allow for measurements at the single cell level. For short term pHi regulation, cellular buffer capacity and mitochondrial function in situ will be assessed as a function of temperature. For long term pHi regulation, the capacities of Cl-:HCO3-, Na-dependent Cl-:HCO3- and Na+:H+ exchange processes are judged from effects of anion transport inhibitors (disulfonic stilbenes) and inhibitors of Na+ transport (furosemide and amiloride). Evidence for a H+ pump will also be obtained. Regulation of pCai will be examined in terms of mitochondrial function and interrelationships with pHi. Correlations of cell survival with ability of cells to regulate pHi and pCai will be based on the reversibilities of these processes after heat treatment, examination of thermotolerant cells and the effects of the above inhibitors on cell survival. The development of photometric fluorescence microscopy to single cell analysis and its application to hyperthermia studies may allow for the biochemical characterization of thermotolerant cells and cells doomed to die after heat treatment.